MLL3/MLL4 enzymatic activity shapes DNA replication timing.

Mammalian genomes are replicated in a precise order during S phase, which is cell-type-specific1-3 and correlates with local transcriptional activity2,4-8, chromatin modifications9 and chromatin architecture1,10,11,12. However, the causal relationships between these features and the key regulators of DNA replication timing (RT) are largely unknown. Here, machine learning was applied to quantify chromatin features, including epigenetic marks, histone variants and chromatin architectural factors, best predicting local RT under steady-state and RT changes during early embryonic stem (ES) cell differentiation. About one-third of genome exhibited RT changes during the differentiation. Combined, chromatin features predicted steady-state RT and RT changes with high accuracy. Of these features, histone H3 lysine 4 monomethylation (H3K4me1) catalyzed by MLL3/4 (also known as KMT2C/D) emerged as a top predictor. Loss of Mll3/4 (but not Mll3 alone) or their enzymatic activity resulted in erasure of genome-wide RT dynamics during ES cell differentiation. Sites that normally gain H3K4me1 in a MLL3/4-dependent fashion during the transition failed to transition towards earlier RT, often with transcriptional activation unaffected. Further analysis revealed a requirement for MLL3/4 in promoting DNA replication initiation zones through MCM2 recruitment, providing a direct link for its role in regulating RT. Our results uncover MLL3/4-dependent H3K4me1 as a functional regulator of RT and highlight a causal relationship between the epigenome and RT that is largely uncoupled from transcription. These findings uncover a previously unknown role for MLL3/4-dependent chromatin functions which is likely relevant to the numerous diseases associated with MLL3/4 mutations.

Transcriptional repression upon S phase entry protects genome integrity in pluripotent cells.

Coincident transcription and DNA replication causes replication stress and genome instability. Rapidly dividing mouse pluripotent stem cells are highly transcriptionally active and experience elevated replication stress, yet paradoxically maintain genome integrity. Here, we study FOXD3, a transcriptional repressor enriched in pluripotent stem cells, and show that its repression of transcription upon S phase entry is critical to minimizing replication stress and preserving genome integrity. Acutely deleting Foxd3 leads to immediate replication stress, G2/M phase arrest, genome instability and p53-dependent apoptosis. FOXD3 binds near highly transcribed genes during S phase entry, and its loss increases the expression of these genes. Transient inhibition of RNA polymerase II in S phase reduces observed replication stress and cell cycle defects. Loss of FOXD3-interacting histone deacetylases induces replication stress, while transient inhibition of histone acetylation opposes it. These results show how a transcriptional repressor can play a central role in maintaining genome integrity through the transient inhibition of transcription during S phase, enabling faithful DNA replication.